Chagas disease is a tropical parasitic disease caused by the flagellate protozoan Trypanosoma cruzi. T. cruzi is commonly transmitted to humans and other mammals by an insect vector, the blood-sucking “kissing bugs” of the subfamily Triatominae (family Reduviidae; in German: “Raubwanzen”). The disease may also be spread through blood transfusion and organ transplantation, ingestion of food contaminated with parasites, and from a mother to her fetus.
Trypanosoma cruzi appears in different forms and development stages. The reproducing form is called epimastigote which is adapted just after the kissing bug has taken a blood meal on an infected animal including humans. The epimastigotes move onto the rectal cell wall of the bug. The bug transfers the pathogen via its feces to the next host in a subsequent blood meal where the bug defecates. The infectious form is called trypomastigote and enters the human body through the bite wound. The trypomastigote can therefore be found in human blood. A further form found e.g. in the cytoplasma of heart muscle cells is called amastigote or micromastigote. During the life cycle of T. cruzi the amastigote changes to trypomastigote which can be sucked in at the next meal by a kissing bug.
The symptoms of Chagas disease vary over the course of an infection. Often there is an acute phase followed by a chronic phase. There can also be a latent phase after infection. Each phase can be symptom free or life-threatening. In the early, acute stage, symptoms are generally mild and usually produce no more than local swelling at the site of infection. The initial acute phase is responsive to antiparasitic treatments, with 60-90% cure rates. After 4-8 weeks, individuals with active infections enter the chronic phase of Chagas disease that is asymptomatic for 60-80% of chronically infected individuals through their lifetime. During the chronic phase, some patients develop cardiac complications leading to an enlarged heart, heart failure, altered heart rate and sudden death. Also intestinal complications leading to difficulties with eating or passing stool are typical of the chronic stage.
The antiparasitic treatments also appear to delay or prevent the development of disease symptoms during the chronic phase of the disease, but according to the U. S. Centers for Disease Control and Prevention the average life-time risk of developing one or more of these complications is about 30% which means that these chronically infected individuals will still eventually develop life-threatening heart and digestive system disorders. The currently available antiparasitic treatments for Chagas disease are benznidazole and nifurtimox, which can cause temporary side effects in many patients including skin disorders, brain toxicity, and digestive system irritation.
Chagas disease mainly appears in poor, rural areas of Mexico, Central America, and South America; very rarely, the disease has been found in the Southern United States. However, blood donors are screened for infection with Trypanosoma cruzi by in vitro diagnostic methods in these countries.
Today several serologic diagnostic methods are available to detect infections with T. cruzi, e.g. detection of antibodies against T. cruzi by indirect immunofluorescence, indirect hemagglutination, complement fixation, immunoblot techniques and ELISAs. Also methods of molecular biology (e.g. PCR) and elaborate xenodiagnostic methods are applied. In xenodiagnostics a vector-transmitted infection a laboratory-reared, pathogen-free insect (here: the kissing bug) is allowed to suck blood from a patient. The intestinal contents of the insect are then examined for the presence of the pathogen (here: Trypanosoma cruzi).
Each of these methods shows its own weaknesses and strengths with regard to sensitivity and specificity and accordingly there is no gold standard method available so far.
In the beginning of Chagas assay development for detection of antibodies native antigen lysates were applied and are still being used. However, using lysates only one of the three development stages of T. cruzi is represented in this antigen composition so that there is a certain likelihood to miss infections of the two other stages. More modern assays apply mixtures of recombinant antigens, representing all stages of T. cruzi infection.
When native antigen lysates are used the diagnostic assay often faces problems in specificity and cross-reactivities observed in samples of patients that have been infected by Leishmania, another parasite. In addition, the production of antigen lysates leads to considerable lot-to-lot variation because of the complex antigen composition of T. cruzi. Moreover, very often rare reagents base on native lysates show a weak sensitivity as some lysates do not contain at all or do not contain sufficient antigens of all life cycle stages.
By applying recombinant antigens the above challenges can be circumvented or avoided. However, commercially available assay kits for detecting Chagas disease which are based on recombinant antigen compositions show considerable differences with respect to sensitivity and specificity so that customers, i.e. commercial or clinical labs or blood screening units, often have to use several kits in parallel to obtain reliable results. As a consequence a decision on whether a patient's sample is reactive or not is based on a majority of positive or negative results obtained for the same sample by several kits based on different antigen compositions. It is obvious that this time-consuming procedure of applying multiple diagnostic tests does not make sense economically as it leads to an increase of lab equipment and personal, time, workload and costs.
Serological assays for detecting antibodies against Trypanosoma cruzi antigens have been widely described in prior art literature, for review see for example Silveira et al. Trends in Parasitology 2001, Vol. 17 No. 6. T. cruzi recombinant antigens relevant for serodiagnosis have been isolated by several laboratories. Several of these genes have tandemly repeated sequences. Due to the extremely large number of antigenic proteins expressed by Trypanosoma cruzi (approximately 23000 predicted protein coding sequences and pseudogenes in public data bases) the number of possible combinations of antigens for an immunoassay is enormously huge. Although methods for recombinant production have been known for decades it still remains a challenge to find out which antigens are required to set up a diagnostic assay. Choosing suitable antigens for an immunodiagnostic assay one has to bear in mind to consider antigens of all life cycle stages of the pathogen and also apply antigens against which antibodies can be found for all stages of infection (acute, window and chronic phase). At the same time the number of antigens should not exceed about 5 or 10 because of technical considerations (e.g. lack of solubility and stability, unwanted cross-reactions leading to quenching of signals, avoidance of cross-reactivity to e.g. Leishmania) and also economic considerations as each antigen in addition needs to be fully developed, evaluated and produced in large scale. According to Silveria et al. (supra) commercially available assays often use a combination of six or seven different T. cruzi antigens, sometimes also in combination of shorter synthetic peptides derived from T. cruzi full length antigens.
Another approach to provide a highly sensitive diagnostic test is based on a multicomponent assay applying a high number of various kinds of T. cruzi antigens that have been coated individually on separate beads. WO 2009/017736 and U.S. Pat. No. 8,329,411 disclose a device and a method for detecting an infection by Trypanosoma cruzi in a biological sample. This set-up includes 16 different proteins (selected from initially 59 candidate proteins, acting as antigens) that have to be coated individually on labeled beads providing an array-like diagnostic tool. Antibodies, if present in the sample, bind to these coated proteins. Consequently the bound antibodies are detected by binding of a labeled secondary antibody to the sample antibodies. While this procedure makes sense for a research approach the high number of antigens leading to large production costs is too costly to be used as a routine assay in a commercial or clinical laboratory.
In summary, the immunoassays for detecting T. cruzi antibodies in samples from infected individuals known in the art apply a high number of different antigens to achieve high sensitivity and specificity. A true gold standard and economically affordable assay is still not available.
The problem therefore can be seen in providing a diagnostic composition and method that overcomes the disadvantages with respect to reproducibility, sensitivity and specificity of the prior art assays for detecting infections with Trypanosoma cruzi. 
The problem is solved by the current invention as specified in the claims.